Comparison of Column Packings for Isocratic High-Performance Liquid Chromatographic Analysis of Carotenoids Using Non-Aqueous Reversed-Phase

Abstract

Ten different columns are compared for the isocratic non-aqueous reversed-phase separation of carotenoids, using solvent mixtures of ethyl acetate-acetonitrile both with and without 0.1% n-decanol as modifier. Conditions were established for separation of a mixture of alfalfa carotenoids containing mainly neoxanthin, violaxanthin, lutein and β-carotene. The best material for use for rapid isocratic separation of all major components was a high carbon loading non-endcapped material with ODS functionality, although one endcapped C8 material gave similar results. The use of n-decanol as mobile phase modifier is imperative to rapidly condition new columns to give optimum peak shape and definition and system linearity.     

Multiresidue Analysis of Some Insect Growth Regulators by Reversed-Phase High-Performance Liquid Chromatography

Abstract

A general procedure has been developed for the analysis of 8 different insect growth regulators (IGRs) by using reversed-phase high-performance liquid chromatography with gradient solvent systems. The method has been used to identify and separate 8 insect growth regulators from a mixture of the standards. The method has been evaluated with different column conditions and under different solvent systems. Best resolution was obtained by using a double column and methanol/water gradient system.     

High Performance Liquid Chromatography of Uroporphyrin Isomers

Abstract

The separation of uroporphyrin I and III isomers by reversed-phase high performance liquid chromatography on ODS-Hypersil with ammonium acetate buffer-acetonitrile solvent systems is described. The effects of buffer concentration, pH, organic modifier proportion and different organic modifiers on the resolution are studied. The optimum conditions for the separation were 12–13% acetonitrile in 1M ammonium acetate buffer pH 5.10–5.20. The method also separated uroporphyrin I and III from the II isomers but the resolution of uroporphyrin III and IV isomers was not achieved.     

High-Performance Liquid Chromatography of T-RNA

Abstract

Fractionation of amino acid specific tRNAs by descending salt gradient on a reversed-phase high-performance liquid chromatography column is demonstrated. Stationary phase variables such as n-alkyl chain length and silica type are discussed in terms of stability and selectivity. Mobile phase parameters including salt concentration, organic modifier, pH, and temperature are investigated. The concentration of organic modifier to adjust elution profiles can be critical in terms of tRNA retention.     

Determination Of Mexacarbate And Five Of Its Derivatives By High-Performance Liquid Chromatography

Abstract

A simple, sensitive and reliable analytical method has been developed and reported for mexacarbate (4-ditnethylamino-3,5-xylyl N-methylcarbamate) and five of its possible degradation products likely to be found in environmental samples using reversed-phase high-performance liquid chromatography with isocratic and gradient solvent systems. All chromatogram peaks were Identified through comparison to standards. The method has been used to identify and separate the six compounds from a mixture of the standards. It has been evaluated under different column conditions and with different mobile phases. Best resolution of the analytes was obtained by using a gradient solvent system consisting of CH₃CN and H₃O detecting at 200 nm and 30°C using a HP-RP8, 10 μ m, 20 cm 4.6 mm column.     

Evaluation of Mobile and Stationary Phases in Reversed-Phase High-Performance Liquid Chromatography of Conjugated Bile Acids

Abstract

The reversed-phase high-performance liquid chromatographic separation of the ten major conjugated bile acids of man using isocratic conditions is described. Each component of the mobile and stationary phases was examined for its ability to influence the separation selectivity. Manipulation of pH, buffer species, organic modifier and different types of packings showed that optimal resolution was obtained with a mobile phase of methanol-0.02M sodium acetate (60:30) adjusted to pH 4.2 with phosphoric acid, on a Supelcosil LC-18-DB column. Advantages of the optimized phase system are the complete baseline separation of compounds within a short period of time, improved peak symmetry and a high rate of reproducibility. This new chromatographic method, coupled with UV detection at 205 nm, is suitable for the simultaneous determination of bile acid conjugates in routine clinical analysis.     

Dynamic column-coating procedure for chiral ligand-exchange chromatography

Summary The preparation of HPLC columns for chiral ligand-exchange chromatography by dynamic adsorption of a histidine-based chiral selector (N-n-decyl-L-histidine, LNDH) on commercial reversed-phase, C₁₈ columns was investigated. The most suitable solvent for column preparation was sought through a study of the behaviour of LNDH on the reversed-phase column eluting with different methanol-water ratios at varying pH. It is shown that the use of an aqueous solvent instead of pure methanol is advantageous, despite the decrease in LNDH solubility, and that the solvent pH must be controlled. Open and closed-cycle procedures for flowing the selector solution through the column are compared through a study of chromatographic retention, selectivity and efficiency parameters of selected enantiomers. Even though no significant difference in column loading was found between the two procedures, the open cycle method gave rise to more efficient and selective chiral columns.     

LC-amperometric determination of tert-butylcatechol in N-methyl-2-pyrrolidone stream containing polar and nonpolar impurities

Amperometric detection is shown to be highly selective and sensitive for the determination of tert-butylcatechol (TBC) added as an antioxidant in the circulating N-methyl-2-pyrrolidone (NMP) used for the purification of 1,3-butadiene by extractive distillation. The separation of TBC from numerous polar and nonpolar species present in the analyte is achieved on a 5-cm reversed-phase C-18 column using acetonitrile-water gradient conditions, both solvent components containing tetrabutyl ammonium dihydrogen phosphate as the amphiphilic modifier. The separation process is optimized for the resolution of TBC from nonpolar hydrocarbons as well as polar compounds, and a complete elution of all species from the column. An applied potential of 0.6 V relative to the Ag/AgCI electrode is found to provide an optimum sensitivity by the amperometric detector. It is also shown that the hydrocarbon content in NMP can be monitored simultaneously by coupling a spectrophotometric detector in series.     

High-Performance Liquid Chromatography of the Tetradecapeptide Somatostatin

Abstract

Optimum reversed-phase systems for qualitative and quantitative determinations of somatostatin are evaluated. Temperature, pH, buffer concentration, type and concentration of organic modifier and the presence of ion-pairing agents more or less influence the retention and the separating efficiency. The retention behaviour of some analogues is described to illustrate the selectivity of the system. Down to 10-20 ng of somatostatin can be determined, even in the presence of a large excess of albumin, by using 210 nm as the detection wavelength.     

Application of the split-peak effect to study the adsorption kinetics of human serum albumin on a reversed-phase support

The adsorption step of human serum albumin on a reversed-phase support was analyzed by studying the "split-peak" effect in mass-overload conditions. This behavior is characterized by the occurrence of a first non-retained fraction and is described by an analytical expression in the case of a Langmuirian adsorption isotherm. The method was applied to determine the column loading capacity, the number of mass-transfer units and the apparent adsorption rate constant measured at a given flow-rate. The nature of the organic modifier influences the split-peak effect: it increases with the eluotropic strength of the organic solvent added to the buffer. Compared to the results with pure buffer, it is the association of two effects, the decrease of the column loading capacity and that of the apparent adsorption rate constant, which increases the split-peak effects observed when methanol and 2-propanol are added to the eluent. These results allow us to gain a better understanding of the role of the organic solvent in the elution behavior of proteins in reversed-phase high-performance liquid chromatography.     

The Separation of Organic Analyte Cations on a Low-Capacity Cation Exchange Column Using Indirect UV Detection

Abstract

The retention of organic analyte cations on a low-capacity cation exchange column using indirect UV detection was studied. It was found that a combination of cation exchange/reversed-phase interactions affected the retention of organic analyte cations provided the analytes have both a cationic charge site and a hydrophobic center. The factors that influenced the organic analyte cation retention were: concentration of organic modifier, concentration of UV absorbing analyte, pH, and mobile phase ionic strength. Elution orders for several of the organic analytes studied on the low-capacity cation exchange column were different than those observed on silica-based strong cation exchange columns.     

Urinary Uric Acid Determination by Reversed-Phase High-Performance Liquid Chromatography with Electrochemical Detection

Abstract

Determination of urinary uric acid has been attempted by reversed-phase high-performance liquid chromatography with electrochemical detection. We have found that the electrochemical detection method is suitable for monitoring eluate from reversed-phase column and also that the minimum detectable quantity of uric acid using en electrochemical detector is about 10 pg. Complete separation of uric acid was achieved in about 8 min under the present chromatographic conditions.     

Simultaneous Determination of Maprotiline,Desipramine, and Moclobemide by Reversed-Phase High-Performance Liquid Chromatography and Statistical Optimization

Abstract

A method for separation of three antidepressants, maprotiline, desipramine, and moclobemide, by reversed-phase high-performance liquid chromatography (RP-HPLC) was developed and validated. To find optimal conditions and estimate the impact of individual parameters on the separation, a complete set of 2³ interdependent relationships of the mobile phase composition, temperature, and the volume flow rate were examined. Full separation of the investigated components from a laboratory mixture was achieved on a Supelcosil LC-18 (120 mm × 4.6 mm, 5 µm) column, using two solvent systems, 3% ammonium ion in water/ethanol and acetonitrile, and alternating isocratic gradient–isocratic elution modes. Relevance of the proposed method for therapeutic drug monitoring is anticipated.     

Retention behaviour of an homologous series of oligodeoxythymidilic acids using reversed-phase ion-pair chromatography

Summary The retention behaviour of an homologous series of phosphorylated oligodeoxythymidylic acid (pd(T)_5–18) oligonucleotides was studied using reversed-phase ion-pair chromatography with isocratic elution conditions. The effects of temperature, pH, eluent ionic strength, percentage organic modifier, concentration and alkyl chain length of the ion-pairing reagent were investigated. The retention behaviour was generally explicable by current theoretical models of ion-pair chromatography. However, the marked effect of mobile phase pH on the retention of the oligonucleotides was unexpected, and this was ascribed to the presence of ionisable residual silanols on the surface of the reversed-phase packing material.     

Simultaneous Determination of Dopamine,Norepinephrine, Tyramine and Octopamine by Reverse-Phase High Performance Liquid Chromatography with Electrochemical Detection

Abstract

A method is described for the simultaneous separation and estimation of the catecholamines, norepinephrine and dopamine and their monohydroxy-equivalents, octopamine and tyramine. The method employs high-performance liquid chromatographic separation of the compounds on a C18 reverse-phase column with a mobile phase containing methanol as the organic modifier, octane sulphonate as an ion-pair reagent and acetic acid/ammonium hydroxide buffer. The influences of electrode potential and solvent pH on detector response were studied, and the optimal conditions identified as detector potential of 0.95 volts and pH 6.0. The technique of post-column mixing was introduced to provide optimal pH conditions for detector response without the constraint of on-column oxidation of catecholamines. The effects of buffer ionic strength on retention factors and detector response were also investigated and, on the basis of the results obtained, the optimal buffer strength was identified as 0.08–0.09 molar. The described procedure can be used for simultaneous estimation of catecholamines and monohydroxyphenolamines at concentrations between 200–5000 pg.     

Reversed-Phase Systems for the Separation of Coumarins and Furocoumarins by Thin-Layer and High-Performance Liquid Chromatography

Abstract

The retention behaviour of eleven derivatives of counarin was investigated by reversed-phase (RP) thin-layer and high performance liquid chromatography. The linear relationships between log k'or R_M values and the content of organic modifier in the aqueous mobile phase obtained for wide composition ranges indicate that the plots can be used to determine R_Mw and log k_w values by extrapolation to pure water. The effects of individual substituants on the retention and the correlation between TLC and HPLC data was analysed.     

Separation of Carotenoids in Fruits and Vegetables by High Performance Liquid Chromatography

Abstract

Several isocratic high performance liquid chromatographic (HPLC) methods, including both normal and reversed-phase, have been developed to separate provitamin A compounds and other carotenoids - α-carotene, β-carotene, stereoisomers of β-carotene, γ-carotene, β-cryptoxanthin, canthaxanthin and lycopene- in fruits and vegetables. The normal phase systems used either an amino or alumina column with 99.5% isooctane and 0.5% tetrahydrofuran. Procedures employing reversed-phase used C18 columns- Vydac 201TP54, Vydac 218TP54, Zorbax ODS and NovaPak C18- with nonaqueous solvent systems comprised of various mixtures of acetonitrile, methanol, tetrahydrofuran and chloroform. All compounds were monitored at 470 nm or 450 nm. Each method has certain advantages and disadvantages for the analysis of carotenoids in fruits and vegetables.     

Synthesis and characterization of phenylpropanolamine bonded silica for multimode liquid chromatography of small molecules

Summary A multimodal silica support carrying both anionexchange and hydrophobic functions was prepared by bonding phenylpropanolamine to epoxysilane-modified silica. This support was characterized with regard to its physical and chromatographic properties and has a surface coverage of 150 mol g^–1. The reversed-phase and the anion-exchange behaviour of the new stationary phase were investigated by injection of acidic, neutral and basic drugs and inorganic anions. The retention of the tested compounds was manipulated by modifying the pH and proportion of organic modifier in the mobile phase in isocratic mode. The mixed-mechanism column provided a flexible and versatile method for the simultaneous separation of neutral, acidic and basic compounds.     

A Simple and Rapid Reversed Phase High Performance Liquid Chromatographic Method for Quantification of Alprazolam and α-Hydroxyalprazolam in Plasma

Abstract

A simple and rapid reversed-phase liquid chromatographic method for the determination of alprazolam and a-hydroxyalprazolam in plasma is described. Flunictrazepam was used as internal standard. Plasma samples were buffered with sodium borate and extracted with dichloromethane /n-pentane 4:6 v/v for 60 sec on a vortex apparatus. Extraction solvent was evaporated to dryness and extraction residues were reconstituted in the mobile phase. Samples were chromatographed on a 5μ Lichrospher RP-18 column (25cm × 4mm i. d) using acetonitrile/water 40:60 v/v as the mobile phase. The column effluent was monitored at 230nm. The lower limit of detection was 1ng/ml for alprazolam and a-hydroxyalprazolam while the lower limit of quantification was 2ng/ml for both compounds. Peak height and plasma     

Quantitation of Carbon Dioxide by High Performance Liquid Chromatography: The Key to Solubility Measurements in Liquefied Gas Solutions

Abstract

A technique is described whereby solubility data may be obtained for systems employing liquefied carbon dioxide as the solvent. Analysis of the solution is effected by the direct injection of an aliquot of pressurized liquid into a modern liquid chromatograph fitted with a reversed-phase C₁₈ column. Detection of the carbon dioxide is accomplished by a differential refractometer. After the appropriate calibrations are performed, data are obtained which compare favourably with the literature value of the solubility of naphthalene in liquid carbon dioxide.